Abstract: Great advances in our knowledge are often the result of innovative technological advances. For example, combining light microscopy with Golgi staining led to the birth of modern neuroscience. My proposal aims to provide a major leap in the methodology of neuroscience research that holds the potential of transforming future studies of brain function and dysfunction. Brain function relies on tiny specialized structures at the synapse where protein molecules are arranged with nanometer precision. Small changes in synaptic machinery are viewed as early manifestations of many neurological disorders and neurodegenerative diseases, which are increasingly prevalent as the population ages. However, it has been challenging to examine synaptic protein organization at a sufficiently high level of resolution, especially in brain tissue. Surprisingly, this limitation does not lie in the microscopic methods. Fluorescence super-resolution microscopic methods such as the photoactivated localization microscopy (PALM) that can probe with 10-nm resolution have been developed. However, obstacles associated with protein labeling, sample preparation and data interpretation have prevented their application to brain tissue. Herein, I propose to develop innovative methodologies to label endogenous synaptic proteins and prepare brain samples for PALM. We will also establish a novel approach for visualizing the super-resolution protein organization within a cellular context to aid the interpretation of the data. We will do so by combining innovative developments across multiple disciplines, including molecular biology, biochemistry, genetics, super-resolution fluorescence microscopy, electron microscopy and computer programming. If established, our techniques will revolutionize the methodologies used in neuroscience research by providing unprecedented abilities to obtain fine details of synaptic architecture. These methods will be applicable to the study of other cellular proteins. The ability to identify previously undetectable subtle changes will also enable early diagnoses and an enhanced mechanistic understanding of neurological diseases affecting synaptic and other cellular structures. Public Health Relevance: Subtle changes in protein organization in the brain are an early manifestation of many neurological disorders and neurodegenerative diseases, which are increasingly prevalent as the population continues to age. We aim to develop methods that can visualize these previously-undetectable changes to enable early diagnoses and an enhanced mechanistic understanding of neurological diseases affecting synaptic structures.